Identification of a new class of epsp synthases

ABSTRACT

Compositions and methods for conferring tolerance to glyphosate in bacteria, plants, plant cells, tissues and seeds are provided. Compositions include a novel class of EPSPS enzymes, designated Class III, and polynucleotides encoding such enzymes, vectors comprising those polynucleotides, and host cells comprising the vectors. The novel proteins comprise at least one sequence domain selected from the Class III domains provided herein. These sequence domains can be used to identify EPSP synthases with glyphosate resistance activity.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 11/400,598, filed Apr. 7, 2006 which claims the benefit of U.S.Provisional Application Ser. Nos. 60/669,686, filed Apr. 8, 2005;60/678,348 filed May 6, 2005; 60/695,193 filed Jun. 29, 2005; and60/725,182 filed Oct. 11, 2005, the contents of which are herebyincorporated in their entirety by reference herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“384669_SequenceListing.txt”, created on Feb. 15, 2010, and having asize of 132 kilobytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to plant molecular biology, particularly to anovel class of EPSP synthases that confer resistance to the herbicideglyphosate.

BACKGROUND OF THE INVENTION

N-phosphonomethylglycine, commonly referred to as glyphosate, is animportant agronomic chemical. Glyphosate inhibits the enzyme thatconverts phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid (S3P)to 5-enolpyruvyl-3-phosphoshikimic acid. Inhibition of this enzyme(5-enolpyruvylshikimate-3-phosphate synthase; referred to herein as“EPSP synthase”, or “EPSPS”) kills plant cells by shutting down theshikimate pathway, thereby inhibiting aromatic amino acid biosynthesis.

Since glyphosate-class herbicides inhibit aromatic amino acidbiosynthesis, they not only kill plant cells, but are also toxic tobacterial cells. Glyphosate inhibits many bacterial EPSP synthases, andthus is toxic to these bacteria. However, certain bacterial EPSPsynthases have a high tolerance to glyphosate.

Plant cells resistant to glyphosate toxicity can be produced bytransforming plant cells to express glyphosate-resistant bacterial EPSPsynthases. Notably, the bacterial gene from Agrobacterium tumefaciensstrain CP4 has been used to confer herbicide resistance on plant cellsfollowing expression in plants. A mutated EPSP synthase from Salmonellatyphimurium strain CT7 confers glyphosate resistance in bacterial cells,and confers glyphosate resistance on plant cells (U.S. Pat. Nos.4,535,060; 4,769,061; and 5,094,945).

EPSP synthase (Mr 46,000) folds into two similar domains, eachcomprising three copies of a βαβαββ-folding unit (Stallings et al.(1991) Proc. Natl. Acad. Sci. U.S.A. 885046-5050). Lys-22, Arg-124,Asp-313, Arg-344, Arg-386, and Lys-411 are conserved residues of theEPSP synthase from E. coli (Schönbrunn et al. (2001) Proc. Natl. Acad.Sci. U.S.A. 98:1376-1380). Conserved residues important for EPSPSactivity also include Arg-100, Asp-242, and Asp-384 (Selvapandiyan etal. (1995) FEBS Letters 374:253-256). Arg-27 binds to S3P (Shuttleworthet al. (1999) Biochemistry 38:296-302). Variants of the wild-type EPSPSenzyme have been isolated which are glyphosate-tolerant as a result ofalterations in the EPSPS amino acid coding sequence (Kishore and Shah(1988) Annu. Rev. Biochem. 57:627-63; Wang et al. (2003) J. Plant Res.116:455-60; Eschenburg et al. (2002) Planta 216:129-35). He et al.(2001, Biochim et Biophysica Acta 1568:1-6) have developed EPSPsynthases with increased glyphosate tolerance by mutagenesis andrecombination between the E. coli and Salmonella typhimurium EPSPSgenes, and suggest that mutations at position 42 (T42M) and position 230(Q230K) are likely responsible for the observed resistance. Subsequentwork (He et al (2003) Biosci. Biotech. Biochem. 67:1405-1409) shows thatthe T42M mutation (threonine to methionine) is sufficient to improvetolerance of both the E. coli and Salmonella typhimurium enzymes.

Due to the many advantages herbicide resistance plants provide, methodsfor identifying herbicide resistance genes with glyphosate resistanceactivity are desirable.

SUMMARY OF INVENTION

Compositions and methods for conferring tolerance to glyphosate inbacteria, plants, plant cells, tissues and seeds are provided.Compositions include a novel class of EPSPS enzymes, designated ClassIII, and polynucleotides encoding such enzymes, vectors comprising thosepolynucleotides, and host cells comprising the vectors. The novelproteins comprise at least one sequence domain selected from thefollowing domains (Class III domains):

Domain I:

-   L-A-K-G-X₁-S-X₂-L-X₃-G-A-L-K-S-D-D-T (SEQ ID NO:13), where X₁    denotes lysine or threonine, where X₂ denotes arginine or histidine,    where X₃ denotes serine or threonine;

Domain Ia:

-   L-A-K-G-X₁, (SEQ ID No:14), where X₁ denotes lysine or threonine;

Domain Ib:

-   S-X₁-L-X₂ (SEQ ID NO:15), where X₁ denotes arginine or histidine,    where X₂ denotes serine or threonine;

Domain Ic:

-   G-A-L-K-S-D-D-T (SEQ ID NO:16);

Domain II:

-   E-P-D-X₁-X₂-T-F-X₃-V-X₄-X₅-X₆-G (SEQ ID NO:17), where X₁ denotes    aspartic acid or alanine, where X₂ denotes serine or threonine,    where X₃ denotes valine or isoleucine, where X₄ denotes threonine or    glutamic acid or lysine, where X₅ denotes serine or glycine, where    X₆ denotes glutamine or serine or glutamic acid or threonine;

Domain IIa:

-   E-P-D-X₁-X₂-T-F-X₃-V (SEQ ID NO:18), where X₁ denotes aspartic acid    or alanine, where X₂ denotes serine or threonine, where X₃ denotes    valine or isoleucine;

Domain IIb:

-   X₁-X₂-X₃-G (SEQ ID NO:19), where X₁ denotes threonine or glutamic    acid or lysine, where X₂ denotes serine or glycine, where X₃ denotes    glutamine or serine or glutamic acid or threonine;

Domain III:

-   RFLTAA (SEQ ID NO:20);

Domain IV:

-   KRPI(G/M)P (SEQ ID NO:21)-   K-R-P-I-X₁-P, where X₁ denotes glycine or methionine or leucine;

Domain V:

-   X₁-G-C-P-P-V (SEQ ID NO:22), where X₁ denotes threonine or serine;

Domain VI:

-   I-G-A-X₁-G-Y-X₂-D-L-T (SEQ ID NO:23), where X₁ denotes arginine or    lysine or leucine, and where X₂ denotes isoleucine or valine;

Domain VII:

-   W-X₁-V-X₂-X₃-T-G (SEQ ID NO:24), where X₁ denotes arginine or    lysine, where X₂ denotes alanine or histidine or glutamic acid or    serine, where X₃ denotes proline or alanine;

Domain VIII:

-   E-P-D-A-S-A-A-T-Y-L-W-X₁-A-X₂-X₃-L (SEQ ID NO:25), where X₁ denotes    alanine or glycine, where X₂ denotes glutamic acid or glutamine,    where X₃ denotes valine or leucine or alanine;

Domain VIIIa:

-   E-P-D-A-S-A-A-T-Y-L-W (SEQ ID NO:26);

Domain IX:

-   I-D-X₁-G (SEQ ID NO:27), where X₁ denotes isoleucine or leucine;

Domain X:

-   F-X₁-Q-P-D-A-K-A (SEQ ID NO:28), where X₁ denotes threonine or    serine;

Domain XI:

-   X₁-F-P-X₂-X₃-X₄-A-X₅-X₆-X₇-G-S-Q-M-Q-D-A-I-P-T-X₈-A-V-X₉A-A-F-N (SEQ    ID NO:29), where X₁ denotes glutamine or lysine or serine, where X₂    denotes asparagine or histidine, where X₃ denotes methionine or    leucine, where X₄ denotes proline or glutamine, where X₅ denotes    threonine or glutamic acid or valine, where X₆ denotes valine or    isoleucine, where X₇ denotes aspartic acid or valine, where X₈    denotes leucine or isoleucine, where X₉ denotes leucine or    isoleucine;

Domain XIa:

-   X₁-F-P-X₂-X₃-X₄-A (SEQ ID NO:30), where X₁ denotes glutamine or    lysine or serine, where X₂ denotes asparagine or histidine, where X₃    denotes methionine or leucine, where X₄ denotes proline or    glutamine;

Domain XIb:

-   X₁-X₂-X₃-G-S-Q-M-Q-D-A-I-P-T-X₄-A-V-X₅A-A-F-N (SEQ ID NO:31), where    X₁ denotes threonine or glutamic acid or valine, where X₂ denotes    valine or isoleucine, where X₃ denotes aspartic acid or valine,    where X₄ denotes leucine or isoleucine, where X₅ denotes leucine or    isoleucine;

Domain XIc:

-   G-S-Q-M-Q-D-A-I-P-T (SEQ ID NO:32);

Domain XII:

-   P-V-R-F-X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:33), where X₁    denotes valine or threonine, where X₂ denotes glutamic acid or    glycine, where X₃ denotes leucine or isoleucine, where X₄ denotes    alanine or glutamic acid, where X₅ denotes isoleucine or valine;

Domain XIIa:

-   P-V-R-F (SEQ ID NO:34);

Domain XIIb:

-   X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:35), where X₁ denotes    valine or threonine, where X₂ denotes glutamic acid or glycine,    where X₃ denotes leucine or isoleucine, where X₄ denotes alanine or    glutamic acid, where X₅ denotes isoleucine or valine;

Domain XIIc:

-   N-L-R-V-K-E-C-D-R (SEQ ID NO:36);

Domain XIII:

-   E-G-D-D-L-X₁-X₂ (SEQ ID NO:37), where X₁ denotes leucine or    isoleucine, where X₂ denotes valine or isoleucine;

Domain XIV:

-   X₁-P-X₂-L-A-G (SEQ ID NO:38), where X₁ denotes aspartic acid or    asparagine, where X₂ denotes alanine or serine or threonine;

Domain XV:

-   A-X₁-I-D-X₂-X₃-X₄-D-H-R (SEQ ID NO:39), where X₁ denotes leucine or    serine or glutamic acid, where X₂ denotes threonine or serine, where    X₃ denotes histidine or phenylalanine, where X₄ denotes alanine or    serine;

Domain XVI:

-   F-A-L-A-X₁-L-K-X₂-X₃-G-I (SEQ ID NO:40), where X₁ denotes glycine or    alanine, where X₂ denotes isoleucine or valine, where X₃ denotes    serine or glycine or alanine or lysine;

Domain XVIa:

-   F-A-L-A-X₁-L-K (SEQ ID NO:41), where X₁ denotes glycine or alanine;

Domain XVIb:

-   L-K-X₁-X₂-G-I (SEQ ID NO:42), where X₁ denotes isoleucine or valine,    where X₂ denotes serine or glycine or alanine or lysine; and

Domain XVII:

-   -X₁-P-X₂-C-V-X₃-K (SEQ ID NO:43), where X₁ denotes asparagine or    aspartic acid, where X₂ denotes alanine or aspartic acid, where X₃    denotes alanine or glycine.

Domain XVIII:

-   X₁-S-L-G-V (SEQ ID NO:44), where X₁ denotes alanine or serine or    proline.

The above domains set forth in SEQ ID NOS:13-44 were identified byaligning Class III sequences that share at least 50% sequence identity.The presence of at least one of these sequence domains is predictive ofglyphosate resistance activity.

Isolated nucleic acid molecules corresponding to herbicideresistance-conferring nucleic acid sequences are provided. Additionally,amino acid sequences corresponding to the polynucleotides areencompassed. In particular, the present invention provides for isolatednucleic acid molecules comprising a Class III domain, including thenucleotide sequence set forth in SEQ ID NOS:9, 11, 55, 57 and 58, anucleotide sequence encoding the amino acid sequence shown in SEQ IDNO:10, 12, 56, and 59, the herbicide resistance nucleotide sequencedeposited in a bacterial host as Accession Nos. B-30833 and B-30838, aswell as variants and fragments thereof. Nucleotide sequences that arecomplementary to a nucleotide sequence of the invention, or thathybridize to a sequence of the invention are also encompassed. Thesequences find use in the construction of expression vectors forsubsequent transformation into plants of interest, as probes for theisolation of other glyphosate resistance genes, as selectable markers,and the like.

Plasmids containing the herbicide resistance nucleotide sequences of theinvention were deposited in the permanent collection of the AgriculturalResearch Service Culture Collection, Northern Regional ResearchLaboratory (NRRL) on Apr. 4, 2005, and assigned Accession No. B-30833.This deposit will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. Access to these deposits will beavailable during the pendency of the application to the Commissioner ofPatents and Trademarks and persons determined by the Commissioner to beentitled thereto upon request. Upon allowance of any claims in theapplication, the Applicants will make available to the public, pursuantto 37 C.F.R. § 1.808, sample(s) of the deposit with the ATCC. Thisdeposit was made merely as a convenience for those of skill in the artand is not an admission that a deposit is required under 35 U.S.C. §112.

Compositions also include antibodies to the polypeptides as well assynthetic polynucleotides encoding herbicide resistance polypeptides.The coding sequences can be used in DNA constructs or expressioncassettes for transformation and expression in organisms, includingmicroorganisms and plants. Compositions also comprise transformedbacteria, plants, plant cells, tissues, and seeds that are glyphosatetolerant by the introduction of the compositions of the invention intothe genome of the organism. Where the organism is a plant, theintroduction of the sequence allows for glyphosate containing herbicidesto be applied to the crop to selectively kill the glyphosate sensitiveweeds, but not the transformed organism.

Methods for identifying an EPSP synthase with glyphosate resistanceactivity are additionally provided. The methods comprise obtaining anamino acid sequence for an EPSP synthase, and identifying whether theamino acid sequence comprises at least one sequence domain of theinvention.

The EPSP synthases described herein represent a new class of EPSPSenzymes, referred to hereinafter as Class III EPSPS enzymes.

DESCRIPTION OF FIGURES

FIG. 1A-C shows an alignment of EPSP synthase amino acid sequences. Theconserved residues that have been identified as important for substratebinding and for EPSPS activity are boxed. The boxes delineate the aminoacid locations of the Class III domains. Roman numerals above the boxescorrespond to the Class III domains.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is drawn to compositions and methods forconferring herbicide tolerance, particularly glyphosate tolerance, inorganisms is provided. The methods involve transforming organisms withnucleotide sequences encoding a Class III glyphosate tolerance gene ofthe invention. In particular, the present invention recognizes a classof enzymes that confers glyphosate tolerance and nucleotide sequencesencoding such enzymes. The sequences find use in preparing plants thatshow increased tolerance to the herbicide glyphosate. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.

The Class III enzymes are characterized by having at least one domainselected from the domains listed below, herein referred to as Class IIIdomains:

Domain I:

-   L-A-K-G-X₁-S-X₂-L-X₃-G-A-L-K-S-D-D-T (SEQ ID NO:13), where X₁    denotes lysine or threonine, where X₂ denotes arginine or histidine,    where X₃ denotes serine or threonine;

Domain Ia:

-   L-A-K-G-X₁, (SEQ ID NO:14), where X₁ denotes lysine or threonine;

Domain Ib:

-   S-X₁-L-X₂ (SEQ ID NO:15), where X₁ denotes arginine or histidine,    where X₂ denotes serine or threonine;

Domain Ic:

-   G-A-L-K-S-D-D-T (SEQ ID NO:16);

Domain II:

-   E-P-D-X₁-X₂-T-F-X₃-V-X₄-X₅-X₆-G (SEQ ID NO:17), where X₁ denotes    aspartic acid or alanine, where X₂ denotes serine or threonine,    where X₃ denotes valine or isoleucine, where X₄ denotes threonine or    glutamic acid or lysine, where X₅ denotes serine or glycine, where    X₆ denotes glutamine or serine or glutamic acid or threonine;

Domain IIa:

-   E-P-D-X₁-X₂-T-F-X₃-V (SEQ ID NO:18), where X₁ denotes aspartic acid    or alanine, where X₂ denotes serine or threonine, where X₃ denotes    valine or isoleucine;

Domain IIb:

-   X₁-X₂-X₃-G (SEQ ID NO:19), where X₁ denotes threonine or glutamic    acid or lysine, where X₂ denotes serine or glycine, where X₃ denotes    glutamine or serine or glutamic acid or threonine;

Domain III:

-   RFLTAA (SEQ ID NO:20);

Domain IV:

-   KRPI(G/M)P (SEQ ID NO:21)-   K-R-P-I-X₁-P, where X₁ denotes glycine or methionine or leucine;

Domain V:

-   X₁-G-C-P-P-V (SEQ ID NO:22), where X₁ denotes threonine or serine;

Domain VI:

-   I-G-A-X₁-G-Y-X₂-D-L-T (SEQ ID NO:23), where X₁ denotes arginine or    lysine or leucine, and where X₂ denotes isoleucine or valine;

Domain VII:

-   W-X₁-V-X₂-X₃-T-G (SEQ ID NO:24), where X₁ denotes arginine or    lysine, where X₂ denotes alanine or histidine or glutamic acid or    serine, where X₃ denotes proline or alanine;

Domain VIII:

-   E-P-D-A-S-A-A-T-Y-L-W-X₁-A-X₂-X₃-L (SEQ ID NO:25), where X₁ denotes    alanine or glycine, where X₂ denotes glutamic acid or glutamine,    where X₃ denotes valine or leucine or alanine;

Domain VIIIa:

-   E-P-D-A-S-A-A-T-Y-L-W (SEQ ID NO:26);

Domain IX:

-   I-D-X₁-G (SEQ ID NO:27), where X₁ denotes isoleucine or leucine;

Domain X:

-   F-X₁-Q-P-D-A-K-A (SEQ ID NO:28), where X₁ denotes threonine or    serine;

Domain XI:

-   X₁-F-P-X₂-X₃-X₄-A-X₅-X₆-X₇-G-S-Q-M-Q-D-A-I-P-T-X₈-A-V-X₉A-A-F-N (SEQ    ID NO:29), where X₁ denotes glutamine or lysine or serine, where X₂    denotes asparagine or histidine, where X₃ denotes methionine or    leucine, where X₄ denotes proline or glutamine, where X₅ denotes    threonine or glutamic acid or valine, where X₆ denotes valine or    isoleucine, where X₇ denotes aspartic acid or valine, where X₈    denotes leucine or isoleucine, where X₉ denotes leucine or    isoleucine;

Domain XIa:

-   X₁-F-P-X₂-X₃-X₄-A (SEQ ID NO:30), where X₁ denotes glutamine or    lysine or serine, where X₂ denotes asparagine or histidine, where X₃    denotes methionine or leucine, where X₄ denotes proline or    glutamine;

Domain XIb:

-   X₁-X₂-X₃-G-S-Q-M-Q-D-A-I-P-T-X₄-A-V-X₅A-A-F-N (SEQ ID NO:31), where    X₁ denotes threonine or glutamic acid or valine, where X₂ denotes    valine or isoleucine, where X₃ denotes aspartic acid or valine,    where X₄ denotes leucine or isoleucine, where X₅ denotes leucine or    isoleucine;

Domain XIc:

-   G-S-Q-M-Q-D-A-I-P-T (SEQ ID NO:32);

Domain XII:

-   P-V-R-F-X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:33), where X₁    denotes valine or threonine, where X₂ denotes glutamic acid or    glycine, where X₃ denotes leucine or isoleucine, where X₄ denotes    alanine or glutamic acid, where X₅ denotes isoleucine or valine;

Domain XIIa:

-   P-V-R-F (SEQ ID NO:34);

Domain XIIb:

-   X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:35), where X₁ denotes    valine or threonine, where X₂ denotes glutamic acid or glycine,    where X₃ denotes leucine or isoleucine, where X₄ denotes alanine or    glutamic acid, where X₅ denotes isoleucine or valine;

Domain XIIc:

-   N-L-R-V-K-E-C-D-R (SEQ ID NO:36);

Domain XIII:

-   E-G-D-D-L-X₁-X₂ (SEQ ID NO:37), where X₁ denotes leucine or    isoleucine, where X₂ denotes valine or isoleucine;

Domain XIV:

-   X₁-P-X₂-L-A-G (SEQ ID NO:38), where X₁ denotes aspartic acid or    asparagine, where X₂ denotes alanine or serine or threonine;

Domain XV:

-   A-X₁-I-D-X₂-X₃-X₄-D-H-R (SEQ ID NO:39), where X₁ denotes leucine or    serine or glutamic acid, where X₂ denotes threonine or serine, where    X₃ denotes histidine or phenylalanine, where X₄ denotes alanine or    serine;

Domain XVI:

-   F-A-L-A-X₁-L-K-X₂-X₃-G-I (SEQ ID NO:40), where X₁ denotes glycine or    alanine, where X₂ denotes isoleucine or valine, where X₃ denotes    serine or glycine or alanine or lysine;

Domain XVIa:

-   F-A-L-A-X₁-L-K (SEQ ID NO:41), where X₁ denotes glycine or alanine;

Domain XVIb:

-   L-K-X₁-X₂-G-I (SEQ ID NO:42), where X₁ denotes isoleucine or valine,    where X₂ denotes serine or glycine or alanine or lysine; and

Domain XVII:

-   -X₁-P-X₂-C-V-X₃-K (SEQ ID NO:43), where X₁ denotes asparagine or    aspartic acid, where X₂ denotes alanine or aspartic acid, where X₃    denotes alanine or glycine.

Domain XVIII:

-   X₁-S-L-G-V (SEQ ID NO:44), where X₁ denotes alanine or serine or    proline.

The above domains set forth in SEQ ID NOS:13-44 were identified byaligning Class III sequences that share at least 50% sequence identity.In some embodiments at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or32 of these sequence domains are present.

Using the methods of the invention and the identified domains,additional proteins (for example, SEQ ID NOS:2, 4, 46 and 48) whichconfer glyphosate tolerance can be identified. These proteins includeknown proteins as well as newly identified proteins (for example, SEQ IDNOS:10, 12, 56, and 59).

By “glyphosate” is intended any herbicidal form ofN-phosphonomethylglycine (including any salt thereof) and other formsthat result in the production of the glyphosate anion in planta. An“herbicide resistance protein,” “herbicide tolerant protein,” or aprotein resulting from expression of an “herbicide resistance-” or“herbicide tolerance-” encoding polynucleotide includes proteins thatconfer upon a cell the ability to tolerate a higher concentration of anherbicide than cells that do not express the protein, or to tolerate acertain concentration of an herbicide for a longer period of time thancells that do not express the protein. A “glyphosate resistance protein”or a “glyphosate tolerant protein” includes a protein that confers upona cell the ability to tolerate a higher concentration of glyphosate thancells that do not express the protein, or to tolerate a certainconcentration of glyphosate for a longer time than cells that do notexpress the protein. By “tolerate” or “tolerance” is intended either tosurvive, or to carry out essential cellular functions such as proteinsynthesis and respiration in a manner that is not readily discernablefrom untreated cells.

Isolated Polynucleotides, and Variants and Fragments thereof

One aspect of the invention pertains to isolated polynucleotides otherthan the polynucleotide sequences listed in SEQ ID NOS:1, 3, 7, 45, 47,and 53 encoding EPSP synthase enzymes having at least one Class IIIsequence domain of the invention. By “other than” is intended that theinvention does not include the polynucleotide sequences set forth in therecited SEQ ID NOS.

The isolated polynucleotides of the present invention comprisenucleotide sequences encoding herbicide resistance proteins andpolypeptides or biologically active portions thereof, as well aspolynucleotides sufficient for use as hybridization probes to identifyherbicide resistance-encoding polynucleotides. As used herein, the term“polynucleotide” is intended to include DNA molecules (e.g., cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The polynucleotide can besingle-stranded or double-stranded DNA.

Nucleotide sequences of the invention include those characterized by thedomains included above. The information used in identifying thesedomains include sequence alignments of glyphosate-sensitive EPSPSmolecules. The sequence alignments were used to identify regions ofhomology between the sequences and to identify the Class III domainsthat are characteristic of Class III EPSPS enzymes.

Variants of the domains are also encompassed within the scope of theinvention (for example SEQ ID NO:55). Such variants include sequencessharing at least 90%, at least 95%, 96%, 97%, 98% or 99% sequenceidentity and are contained in a DNA molecule that imparts glyphosatetolerance.

An “isolated” or “purified” polynucleotide or protein, or biologicallyactive portion thereof, is substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. Preferably, an “isolated” polynucleotide is freeof sequences (for example, protein encoding sequences) that naturallyflank the polynucleotide (i.e., sequences located at the 5′ and 3′ endsof the polynucleotide) in the genomic DNA of the organism from which thepolynucleotide is derived. For purposes of the invention, “isolated”when used to refer to polynucleotides excludes isolated chromosomes. Forexample, in various embodiments, the isolated glyphosateresistance-encoding polynucleotide can contain less than about 5 kb, 4kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence thatnaturally flanks the polynucleotide in genomic DNA of the cell fromwhich the polynucleotide is derived. An herbicide resistance proteinthat is substantially free of cellular material includes preparations ofprotein having less than about 30%, 20%, 10%, or 5% (by dry weight) ofnon-herbicide resistance protein (also referred to herein as a“contaminating protein”).

Polynucleotides that are fragments of these herbicideresistance-encoding nucleotide sequences are also encompassed by thepresent invention (for example, SEQ ID NOS:57, 58, and 60). By“fragment” is intended a portion of the nucleotide sequence encoding anherbicide resistance protein (for example, SEQ ID NO:59). A fragment ofa nucleotide sequence may encode a biologically active portion of anherbicide resistance protein, or it may be a fragment that can be usedas a hybridization probe or PCR primer using methods disclosed below.Polynucleotides that are fragments of an herbicide resistance nucleotidesequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,1700, 1750, 1800, 1850, 1900, 1950 contiguous nucleotides, or up to thenumber of nucleotides present in a full-length herbicideresistance-encoding nucleotide sequence disclosed herein. By“contiguous” nucleotides is intended nucleotide residues that areimmediately adjacent to one another.

Fragments of the nucleotide sequences of the present invention generallywill comprise at least one of the Class III domains described herein,and will encode protein fragments that retain the biological activity ofthe full-length glyphosate resistance protein; i.e.,herbicide-resistance activity. By “retains herbicide resistanceactivity” is intended that the fragment will have at least about 30%, atleast about 50%, at least about 70%, or at least about 80% of theherbicide resistance activity of the full-length glyphosate resistanceprotein disclosed herein as SEQ ID NO:6. Methods for measuring herbicideresistance activity are well known in the art. See, for example, U.S.Pat. Nos. 4,535,060, and 5,188,642, each of which are hereinincorporated by reference in their entirety.

A fragment of an herbicide resistance-encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400 contiguous amino acids, or up to the total number of aminoacids present in a full-length herbicide resistance protein of theinvention. Importantly, the fragment will comprise at least one of theClass III domains described herein.

Herbicide resistance proteins of the present invention are thosecharacterized as Class III or fragments or variants thereof that retainactivity. The term “sufficiently identical” is intended an amino acid ornucleotide sequence that has at least about 60% or 65% sequenceidentity, at least about 70% or 75% sequence identity, at least about80% or 85% sequence identity, or at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% sequence identity compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

To determine the percent identity of two amino acid sequences or of twopolynucleotides, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into theBLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol.215:403-410. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to GDC-like polynucleotides of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to herbicideresistance protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-2402.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. See www.ncbi.nlm.nih.gov. Anothernon-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the ClustalW algorithm (Higgins et al. (1994)Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and alignsthe entirety of the amino acid or DNA sequence, and thus can providedata about the sequence conservation of the entire amino acid sequence.The ClustalW algorithm is used in several commercially availableDNA/amino acid analysis software packages, such as the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, Calif.).After alignment of amino acid sequences with ClustalW, the percent aminoacid identity can be assessed. A non-limiting example of a softwareprogram useful for analysis of ClustalW alignments is GENEDOC™ GENEDOC™(Karl Nicholas) allows assessment of amino acid (or DNA) similarity andidentity between multiple proteins. Another non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithmis incorporated into the ALIGN program (version 2.0), which is part ofthe GCG sequence alignment software package (available from Accelrys,Inc., San Diego, Calif.). When utilizing the ALIGN program for comparingamino acid sequences, a PAM120 weight residue table, a gap lengthpenalty of 12, and a gap penalty of 4 can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

The invention also encompasses variant polynucleotides. “Variants” ofthe herbicide resistance-encoding nucleotide sequences include thosesequences that encode the herbicide resistance protein disclosed hereinbut that differ conservatively because of the degeneracy of the geneticcode, as well as those that are sufficiently identical as discussedabove. Naturally occurring allelic variants can be identified with theuse of well-known molecular biology techniques, such as polymerase chainreaction (PCR) and hybridization techniques as outlined below. Variantnucleotide sequences also include synthetically derived nucleotidesequences that have been generated, for example, by using site-directedmutagenesis but which still encode the herbicide resistance proteinsdisclosed in the present invention as discussed below. Variant proteinsencompassed by the present invention are biologically active, that isthey retain the desired biological activity of the native protein, thatis, herbicide resistance activity. By “retains herbicide resistanceactivity” is intended that the variant will have at least about 30%, atleast about 50%, at least about 70%, or at least about 80% of theherbicide resistance activity of the native protein. Methods formeasuring herbicide resistance activity are well known in the art. See,for example, U.S. Pat. Nos. 4,535,060, and 5,188,642, each of which areherein incorporated by reference in their entirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedherbicide resistance proteins, without altering the biological activityof the proteins. Thus, variant isolated polynucleotides can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore predicted, nonessential amino acid residues. A “nonessential” aminoacid residue is a residue that can be altered from the wild-typesequence of an herbicide resistance protein without altering thebiological activity, whereas an “essential” amino acid residue isrequired for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Amino acid substitutions may bemade in nonconserved regions that retain function. In general, suchsubstitutions would not be made for conserved amino acid residues, orfor amino acid residues residing within a conserved motif, where suchresidues are essential for protein activity. However, one of skill inthe art would understand that functional variants may have minorconserved or nonconserved alterations in the conserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forthe ability to confer herbicide resistance activity to identify mutantsthat retain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingherbicide resistance sequences can be identified by looking for theconserved domains of the invention. See, for example, Sambrook andRussell (2001) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.) and

Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications(Academic Press, St Louis, Mo.).

In a hybridization method, all or part of the herbicide resistancenucleotide sequence or a domain can be used to screen cDNA or genomiclibraries. Methods for construction of such cDNA and genomic librariesare generally known in the art and are disclosed in Sambrook andRussell, 2001, supra. The so-called hybridization probes may be genomicDNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides,and may be labeled with a detectable group such as ³²P, or any otherdetectable marker, such as other radioisotopes, a fluorescent compound,an enzyme, or an enzyme co-factor. Probes for hybridization can be madeby labeling synthetic oligonucleotides based on the known herbicideresistance-encoding nucleotide sequence disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, at least about 25, or at least about 50, 75, 100, 125,150, 175, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1200,1400, 1600, 1800 consecutive nucleotides of herbicideresistance-encoding nucleotide sequence of the invention or a fragmentor variant thereof. Methods for the preparation of probes forhybridization are generally known in the art and are disclosed inSambrook and Russell, 2001, supra and Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), both of which are herein incorporatedby reference.

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length, orless than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of polynucleotides is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments thereof

Herbicide resistance proteins are also encompassed within the presentinvention. By “herbicide resistance protein” is intended a Class IIIprotein having at least one of the domains set forth above, including,for example, SEQ ID NOS:10, 12, 56, and 59. Fragments, biologicallyactive portions, and variants thereof are also provided, and may be usedto practice the methods of the present invention.

“Fragments” or “biologically active portions” include polypeptidefragments comprising a portion of an amino acid sequence encoding anherbicide resistance protein and that retains herbicide resistanceactivity. A biologically active portion of an herbicide resistanceprotein can be a polypeptide that is, for example, 10, 25, 50, 100 ormore amino acids in length. Such biologically active portions can beprepared by recombinant techniques and evaluated for herbicideresistance activity. Methods for measuring herbicide resistance activityare well known in the art. See, for example, U.S. Pat. Nos. 4,535,060,and 5,188,642, each of which are herein incorporated by reference intheir entirety.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, or about at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identical to a Class III enzyme. Variants also include polypeptidesencoded by a polynucleotide that hybridizes to the polynucleotideencoding a Class III enzyme, or a complement thereof, under stringentconditions. Variants include polypeptides that differ in amino acidsequence due to mutagenesis. Variant proteins encompassed by the presentinvention are biologically active, that is they continue to possess thedesired biological activity of the native protein, that is, retainingherbicide resistance activity. Methods for measuring herbicideresistance activity are well known in the art. See, for example, U.S.Pat. Nos. 4,535,060, and 5,188,642, each of which are hereinincorporated by reference in their entirety.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. Furthermore, it isnot often determined a priori which of these codons are used naturallyin the bacterium. Thus, it is understood that use of one of thealternate methionine codons may lead to generation of variants thatconfer herbicide resistance (for example, SEQ ID NO:59 encoded by SEQ IDNO:58). These herbicide resistance proteins are encompassed in thepresent invention and may be used in the methods of the presentinvention.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Transformation of Bacterial or Plant Cells

Transformation of bacterial cells is accomplished by one of severaltechniques known in the art, not limited to electroporation, or chemicaltransformation (See, for example, Ausubel (ed.) (1994) Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., Indianapolis, Ind.).Markers conferring resistance to toxic substances are useful inidentifying transformed cells (having taken up and expressed the testDNA) from non-transformed cells (those not containing or not expressingthe test DNA). In one aspect of the invention, genes are useful as amarker to assess transformation of bacterial or plant cells.

Transformation of plant cells can be accomplished in similar fashion. By“plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen). “Transgenic plants” or “transformed plants” or“stably transformed” plants or cells or tissues refer to plants thathave incorporated or integrated exogenous polynucleotide sequences orDNA fragments into the plant cell. By “stable transformation” isintended that the nucleotide construct introduced into a plantintegrates into the genome of the plant and is capable of beinginherited by progeny thereof.

The genes of the invention may be modified to obtain or enhanceexpression in plant cells. The herbicide resistance sequences of theinvention may be provided in expression cassettes for expression in theplant of interest. “Plant expression cassette” includes DNA constructsthat are capable of resulting in the expression of a protein from anopen reading frame in a plant cell. The cassette will include in the5′-3′ direction of transcription, a transcriptional initiation region(i.e., promoter) operably-linked to a DNA sequence of the invention, anda transcriptional and translational termination region (i.e.,termination region) functional in plants. In some embodiments, thetranscriptional initiation region will cause the production of an RNAsequence that allows for sufficient expression of the encoded EPSPSenzyme to enhance the glyphosate tolerance of a plant cell transformedwith the polynucleotide. By “sufficient expression” is intended that thetranscription initiation region will provide for the expression of anamount of the glyphosate-resistant polypeptide of the invention (e.g.,those containing a Class III domain) that will confer upon a plant orcell the ability to tolerate a higher concentration of glyphosate thanplants or cells that do not contain or express the protein, or totolerate a certain concentration of glyphosate for a longer time thanplants or cells that do not contain or express the protein.

The cassette may additionally contain at least one additional gene to becotransformed into the organism, such as a selectable marker gene.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes. Such an expression cassette is provided with aplurality of restriction sites for insertion of the herbicide resistancesequence to be under the transcriptional regulation of the regulatoryregions.

The promoter may be native or analogous, or foreign or heterologous, tothe plant host and/or to the DNA sequence of the invention.Additionally, the promoter may be the natural sequence or alternativelya synthetic sequence. Where the promoter is “native” or “homologous” tothe plant host, it is intended that the promoter is found in the nativeplant into which the promoter is introduced. Where the promoter is“foreign” or “heterologous” to the DNA sequence of the invention, it isintended that the promoter is not the native or naturally occurringpromoter for the operably linked DNA sequence of the invention.“Heterologous” generally refers to the polynucleotide sequences that arenot endogenous to the cell or part of the native genome in which theyare present, and have been added to the cell by infection, transfection,microinjection, electroporation, microprojection, or the like. By“operably linked” is intended a functional linkage between a promoterand a second sequence, wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. Generally, operably linked means that the polynucleotidesequences being linked are contiguous and, where necessary to join twoprotein coding regions, contiguous and in the same reading frame.

Often, such constructs will also contain 5′ and 3′ untranslated regions.Such constructs may contain a “signal sequence” or “leader sequence” tofacilitate co-translational or post-translational transport of thepeptide of interest to certain intracellular structures such as thechloroplast (or other plastid), endoplasmic reticulum, or Golgiapparatus, or to be secreted. For example, the gene can be engineered tocontain a signal peptide to facilitate transfer of the peptide to theendoplasmic reticulum. By “signal sequence” is intended a sequence thatis known or suspected to result in cotranslational or post-translationalpeptide transport across the cell membrane. In eukaryotes, thistypically involves secretion into the Golgi apparatus, with someresulting glycosylation. By “leader sequence” is intended any sequencethat when translated, results in an amino acid sequence sufficient totrigger co-translational transport of the peptide chain to asub-cellular organelle. Thus, this includes leader sequences targetingtransport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. The plant expression cassette can also beengineered to contain an intron, such that mRNA processing of the intronis required for expression.

By “3′ untranslated region” is intended a nucleotide sequence locateddownstream of a coding sequence. Polyadenylation signal sequences (forexample, polyadenylated nucleotides) and other sequences encodingregulatory signals capable of affecting the addition of polyadenylicacid tracts to the 3′ end of the mRNA precursor are 3′ untranslatedregions. By “5′ untranslated region” is intended a nucleotide sequencelocated upstream of a coding sequence.

Other upstream or downstream untranslated elements include enhancers.Enhancers are nucleotide sequences that act to increase the expressionof a promoter region. Enhancers are well known in the art and include,but are not limited to, the SV40 enhancer region and the 35S enhancerelement.

The termination region may be native with the transcriptional initiationregion, may be native with the herbicide resistance sequence of thepresent invention, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are known in the artfor synthesizing host-preferred genes. See, for example, U.S. Pat. Nos.6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. PublishedApplication Nos. 20040005600 and 20010003849, and Murray et al. (1989)Nucleic Acids Res. 17:477-498, herein incorporated by reference.

In one embodiment, the polynucleotides of interest are targeted to thechloroplast for expression. In this manner, where the polynucleotide ofinterest is not directly inserted into the chloroplast, the expressioncassette will additionally contain a polynucleotide encoding a transitpeptide to direct the gene product of interest to the chloroplasts. Suchtransit peptides are known in the art. See, for example, Von Heijne etal. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol.Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol.84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun.196:1414-1421; and Shah et al. (1986) Science 233:478-481. In someembodiments, the polynucleotides of the invention encode a fusionpolypeptide comprising an amino-terminal chloroplast transit peptide andthe

EPSPS enzyme. A “fusion polypeptide” can be generated, for example, byremoving the stop codon from the polynucleotide sequence encoding afirst polypeptide, then appending the polynucleotide sequence encoding asecond polypeptide in frame such that the resulting polynucleotidesequence will then be expressed by a cell as a single polypeptide.

The polynucleotides of interest to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the polynucleotides of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector.” By “transformation vector” is intended aDNA molecule that is necessary for efficient transformation of a cell.Such a molecule may consist of one or more expression cassettes, and maybe organized into more than one “vector” DNA molecule. For example,binary vectors are plant transformation vectors that utilize twonon-contiguous DNA vectors to encode all requisite cis- and trans-actingfunctions for transformation of plant cells (Hellens and Mullineaux(2000) Trends in Plant Science 5:446-451). “Vector” refers to apolynucleotide construct designed for transfer between different hostcells. “Expression vector” refers to a vector that has the ability toincorporate, integrate and express heterologous DNA sequences orfragments in a foreign cell.

This plant transformation vector may be comprised of one or more DNAvectors needed for achieving plant transformation. For example, it is acommon practice in the art to utilize plant transformation vectors thatare comprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors.” Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the gene of interest are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as in understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science, 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene and in this case“glyphosate”) to recover the transformed plant cells from a group ofuntransformed cell mass. In such processes, glyphosate-resistantpolypeptides comprising one or more Class III domains of the presentinvention may be used as selectable marker.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent (e.g.“glyphosate”). The shoots are then transferred to a selective rootingmedium for recovering rooted shoot or plantlet. The transgenic plantletthen grow into mature plant and produce fertile seeds (e.g. Hiei et al.(1994) The Plant Journal 6:271-282; Ishida et al. (1996) NatureBiotechnology 14:745-750). Explants are typically transferred to a freshsupply of the same medium and cultured routinely. A general descriptionof the techniques and methods for generating transgenic plants are foundin Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239and Bommineni and Jauhar (1997) Maydica 42:107-120. Since thetransformed material contains many cells, both transformed andnon-transformed cells are present in any piece of subjected targetcallus or tissue or group of cells. The ability to kill non-transformedcells and allow transformed cells to proliferate results in transformedplant cultures. Often, the ability to remove non-transformed cells is alimitation to rapid recovery of transformed plant cells and successfulgeneration of transgenic plants. Then molecular and biochemical methodswill be used for confirming the presence of the integrated heterologousgene of interest in the genome of transgenic plant.

Generation of transgenic plants may be performed by one of severalmethods, including but not limited to introduction of heterologous DNAby Agrobacterium into plant cells (Agrobacterium-mediatedtransformation), bombardment of plant cells with heterologous foreignDNA adhered to particles, and various other non-particle direct-mediatedmethods (e.g. Hiei et al. (1994) The Plant Journal 6:271-282; Ishida etal. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994)Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar(1997) Maydica 42:107-120) to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Plants

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

This invention is particularly suitable for any member of the monocotplant family including, but not limited to, maize, rice, barley, oats,wheat, sorghum, rye, sugarcane, pineapple, yams, onion, banana, coconut,and dates.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of the heterologous gene(s) in the plantgenome is confirmed by various methods such as analysis ofpolynucleotides, proteins and metabolites associated with the integratedgene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of the incorporated gene(s) at the earlier stagebefore transplanting into the soil (Sambrook and Russell (2001)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.). PCR is carried out usingoligonucleotide primers specific to the gene of interest orAgrobacterium vector background, etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” then is probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene in the plant genome according to standardtechniques (Sambrook and Russell, 2001, supra).

In Northern analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the sequence of the invention is then tested by hybridizingthe filter to a radioactive probe derived from a GDC, by methods knownin the art (Sambrook and Russell, 2001, supra)

Western blot and biochemical assays and the like may be carried out onthe transgenic plants to determine the presence of protein encoded bythe herbicide resistance gene by standard procedures (Sambrook andRussell, 2001, supra) using antibodies that bind to one or more epitopespresent on the herbicide resistance protein.

The present invention is also drawn to methods for identifying an EPSPsynthase with glyphosate resistance activity. The methods involvedetermining whether certain conserved sequence domains are present inthe amino acid sequence of an EPSP synthase. The presence of one or moreof the domains listed above.

Predicting Protein Function from Sequence

Using the methods of the invention and the identified domains,additional polypeptides (for example, SEQ ID NOS:2, 4, 46 and 48) whichconfer glyphosate tolerance can be identified. These additionalpolypeptides can be identified by searching sequence databasescontaining EPSP synthase sequences, and/or by alignment of polypeptidesequences of EPSP synthase to search for the presence of Class IIIdomains. These polypeptides include known polypeptides as well as newlyidentified polypeptides. It is understood that some modification ofthese domains is tolerated in nature without disrupting the glyphosateresistance conferring nature of these domains, and are thereforeequivalent to the domains listed herein.

In general, there are four levels of protein structure: the primarystructure, which consists of the linear chain of amino acids, or thepolypeptide sequence; the secondary structure, which is given by theα-helices, β-strands, and turns that the protein folds into; thetertiary structure, which is made up of simple motifs that have combinedto form compact globular domains; and the quaternary structure, whichcan comprise several amino acid chains or subunits. When predictingfunction from sequence, it is important to identify the functionallyimportant motifs or patterns. Protein domains with similar folds oftenshare the same molecular function (Hegyi and Gerstein (1999) J. Mol.Biol. 288:147-164; Moult and Melamud (2000) Curr. Opin. Struct. Biol.10:384-389; Shakhnovich et al. (2003) J. Mol. Biol. 326:1-9).Identification of domains important to protein function can be done bymultiple sequence alignment.

Three-dimensional structure can be predicted by homology modeling, i.e.,by using a sequence homologue (>25% sequence identity) with anexperimentally determined 3D structure. The three-dimensional structureof, for example, E. coli EPSP synthase (AroA) is well known (Shönbrunnet al. (2001) Proc. Natl. Acad. Sci. USA 98:1375-1380). This structureis based on the crystallization of AroA with glyphosate and shikimate3-phosphate.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental Example 1 Isolation of EPSPS Genes

Genes coding for class III EPSPS enzymes have been isolated from sevendifferent bacteria (Klebsiella pneumoniae, Agrobacterium radiobacter,Rhizobium sp., Brevundomonas vesicularis, Agrobacterium tumefaciens,Pseudomonas syringae, and Brucella/Ochrobactrum).

The DNA coding sequence and the amino acid sequence of the grg8 openreading frame are provided in U.S. Patent Application No. 60/640,195,filed Dec. 29, 2004, and provided in SEQ ID NO:7 and SEQ ID NO:8 of thisapplication, respectively.

The DNA coding sequence and amino acid sequence of the grg12 openreading frame are provided in SEQ ID NOS:57 and 10 and SEQ ID NOS:58 and59 of this application, respectively.

The DNA coding sequence and amino acid sequence of the grg 15 openreading frame are provided in SEQ ID NO:11 and SEQ ID NO:12 of thisapplication, respectively.

The DNA coding sequence and amino acid sequence of the grg6 open readingframe are provided in GenBank Accession Number AE016853, bases 1,140,091through 1,141,347, and provided in SEQ ID NO:1 and SEQ ID NO:2 of thisapplication, respectively.

The DNA coding sequence and amino acid sequence of the grg9 open readingframe are provided in GenBank Accession Number NC_(—)003304 bases628,398 through 629,675, and provided in SEQ ID NO:3 and SEQ ID NO:4 ofthis application, respectively.

The DNA coding sequence and amino acid sequence of the grg7 open readingframe are provided in SEQ ID NO:45, and SEQ ID NO:46 of thisapplication, respectively.

The DNA coding sequence and amino acid sequence of the grg5 open readingframe are provided in GenBank Accession Number NC_(—)005773, bases 1through 1257, and provided in SEQ ID NO:47 and SEQ ID NO:48 of thisapplication, respectively.

The DNA coding sequence and amino acid sequence of the maize EPSPS openreading frame are provided in GenBank Accession Number X63374(gi:1524383), bases 1 through 1335, and the protein sequence is providedin SEQ ID NO:50 of this application.

The DNA coding sequence and amino acid sequence of a bacterial EPSPSdisclosed in International Patent Application WO2005014820 are providedin SEQ ID NO:53 and SEQ ID NO:54 of this application, respectively.

The DNA coding sequence and amino acid sequence of the grg7m1 openreading frame are provided in SEQ ID NO:55 and SEQ ID NO:56 of thisapplication, respectively.

Example 2 Cloning the EPSP Synthase Gene from Pseudomonas syringae pvTomato Strain DC3000

The EPSP synthase coding sequence was PCR-amplified from genomic DNA ofPseudomonas syringae pv. tomato strain DC3000 (ATCC BAA-871) using thefollowing primers: CAGAGATCTGGCATGCGACCTCAAGCCACTCTC (upper, SEQ IDNO:61) and CAGGGCGCGCCTCAGCGCTGAACACTCACCC (lower, SEQ ID

NO:62). The resultant 1.3 kb PCR product was digested with Bgl II andAsc I, ligated into modified pUC18 which had been digested with BamH Iand Asc I, then electroporated into DH5α cells. Plasmid DNA was preparedfrom ampicillin resistant colonies and analyzed by restriction digest.One clone was chosen for further analysis. The DNA sequence of theinsert was determined using techniques well known in the art and foundto be 100% identical to the published sequence for strain DC3000(Genbank accession number AE016853 bases 1,140,091 through 1,141,347).This plasmid was named pAX703, and the EPSPS ORF was named grg6.

Plasmid pAX703 was transformed into ΔaroA E. coli cells and found tocomplement the deletion. This demonstrated that grg6 encodes afunctional EPSP synthase.

Example 3 Cloning the EPSP Synthase Gene from Agrobacterium radiobacterStrain C58

The EPSP synthase coding sequence was PCR-amplified from genomic DNA ofAgrobacterium tumefaciens strain C58 (ATCC 33970) using the followingprimers: CAGGGATCCGGCATGATCGAACTGACCATCACCC (upper, SEQ ID NO:63) andCAGGGCGCGCCTCAGTGCTGCGGCTCGGCAGCG (lower, SEQ ID NO:64). The resultant1.3 kb PCR product was digested with BamH I and Asc I, ligated intomodified pUC18 which had been digested with BamH I and Asc I, thenelectroporated into DH5α cells. Plasmid DNA was prepared from ampicillinresistant colonies and analyzed by restriction digest. One clone waschosen for further analysis. The DNA sequence of the insert wasdetermined using techniques well known in the art and found to be 100%identical to the published sequence for strain C58 (Genbank accessionnumber NC_(—)003304 bases 628398 through 629675). This plasmid was namedpAX702, and the C58 EPSPS ORF was named grg9.

Plasmid pAX702 was transformed into ΔaroA E. coli cells and found tocomplement the deletion. This demonstrated that grg9 encodes afunctional EPSP synthase.

Example 4 Testing grg6 and grg9 for Resistance to Glyphosate

Plasmids pAX703 and pAX702, containing grg6 and grg9, respectively, weretransformed into E. coli cells and streaked onto M63 agar mediumcontaining various concentrations of glyphosate. The vector plasmidpUC18 was used as a glyphosate-sensitive control. The results arepresented in Table 1 below and demonstrate that expression of grg6 orgrg9, confers resistance to high levels of glyphosate.

TABLE 1 Growth of E. coli expressing grg6 or grg9 in the presence ofglyphosate. Glyphosate Concentration Plasmid Gene 0 mM 50 mM 100 mMpUC18 (none) ++ − − pAX703 grg6 ++ ++ ++ pAX702 grg9 ++ ++ ++

Example 5 Cloning the EPSP Synthase Gene from Pseudomonas syringae pvSyringae Strain B728a

The EPSP synthase coding sequence was PCR-amplified from a single wellisolated colony of Pseudomonas syringae pv syringae strain B728a usingthe following primers: CAGGGATCCGGCATGCGACCTCAAGCCACCCTC (upper, SEQ IDNO:65) and CAGGGCGCGCCTCAGCGCTGAACACTCACAC (lower, SEQ ID NO:66). Theresultant 1.26 kb PCR product was digested with appropriate restrictionenzymes and ligated into the vector pRSF1b, then electroporated into E.coli cells. Plasmid DNA was prepared from ampicillin resistant coloniesand analyzed by restriction digest. One clone was chosen for furtheranalysis and designated as pAX1923. The DNA sequence of the open readingframe in pAX1923 was determined and found to be identical to thepublished sequence of the EPSPS from Pseudomonas syringae pv. syringaestrain B728a. Thus, we designated this open reading frame as grg7.

Similarly, a separate 1.26 kb PCR product was digested with BamH I andAsc I, ligated into modified pUC18 which had been digested with BamH Iand Asc I, then electroporated into DH5α cells. Plasmid DNA was preparedfrom ampicillin resistant colonies and analyzed by restriction digest.One clone was chosen for further analysis. The DNA sequence of theinsert of pAX712 was determined using techniques well known in the artand found to contain 3 nucleotide changes when compared to the publishedDNA sequence of Pseudomonas syringae pv syringae strain B728a (GenbankAccession Number NZ_AABP02000003, bases 39,901 through 41,400). Thesethree DNA nucleotide changes result in a protein with one amino acidchange relative to the hypothetical protein encoded by the publishedsequence. The open reading frame from strain B728a encoding an EPSPS asidentified in plasmid pAX712 was designated grg7m1 (SEQ ID NO:55; grg7mlprotein sequence set forth in SEQ ID NO:56).

Example 6 Cloning the EPSP Synthase Gene from Pseudomonas syringae pvPhaseolicola Strain 1448a

Sequence data for the EPSPS of Pseudomonas syringae pv phaseolicolastrain 1448a was obtained from The Institute for Genomic Researchwebsite at www.tigr.org. The EPSP synthase coding sequence ofPseudomonas syringae pv phaseolicola strain 1448a was PCR-amplified fromgenomic DNA of Pseudomonas syringae pv phaseolicola strain 1448a (ATCCBAA-978) using the following primers: CAGGGATCCGGCATGCGACCTCAAGCCACCCTC(upper, SEQ ID NO:67) and AGAGGCGCGCCTCAGCGCTGAACACGCACC (lower, SEQ IDNO:68), designed based on the DNA sequence available from The Institutefor Genomic Research (“TIGR”, personal communication). The resultant1.26 kb PCR product was digested with BamH I and Asc I, ligated intomodified pUC18 which had been digested with BamH I and Asc I, thenelectroporated into DH5α cells. Plasmid DNA was prepared from ampicillinresistant colonies and analyzed by restriction digest. One clone waschosen for further analysis, and designated pAX713. The DNA sequence ofthe insert of pAX713 was determined using techniques well known in theart and found to be 100% identical to the published DNA sequence ofPseudomonas syringae pv. phaseolicola strain 1448a (performed by TheInstitute for Genomic Research (TIGR), and available online inelectronic form at www.tigr.org). This plasmid was named pAX713, and theopen reading frame from strain 1448a encoding an EPSPS as identified inplasmid pAX713 was designated grg5.

Example 7 Testing of grg5 and grg7 for Resistance to Glyphosate

Plasmids pAX713, pAX1923, and pAX712, containing grg5, grg7, and grg7m1,respectively, were transformed into E. coli cells and streaked onto M63agar medium containing various concentrations of glyphosate. The vectorplasmid pUC18 was used as a glyphosate-sensitive control. The resultsare presented in Table 2 below and demonstrate that expression of grg5,grg7, or grg7m1 confers resistance to high levels of glyphosate.

TABLE 2 Growth of E. coli expressing grg5, grg7, or grg7m1 in thepresence of glyphosate. Glyphosate Concentration Plasmid Gene 0 mM 50 mM100 mM pUC18 (none) ++ − − pAX713 grg5 ++ ++ ++ pAX1923 grg7 ++ ++ ++pAX712 grg7m1 ++ ++ ++

Example 8 Isolation of ATX20019

ATX20019 was isolated by plating samples of soil on HEPES Mineral SaltsMedium (HMSM) containing glyphosate as the sole source of phosphorus.Since HMSM contains no aromatic amino acids, a strain must be resistantto glyphosate in order to grow on this media.

Two grams of soil were suspended in approximately 10 ml of water,vortexed for 15 seconds and permitted to settle for 15 minutes. A 10 μlloopful of this suspension was added to 3 ml of HMSM supplemented with10 mM glyphosate (pH 7.0). HMSM contains (per liter): 10 g glucose, 2 gNH₄SO₄, 9.53 g HEPES, 1.0 ml 0.8 M MgSO₄, 1.0 ml 0.1 M CaCl₂, 1.0 mlTrace Elements Solution (In 100 ml of 1000× solution: 0.1 g FeSO₄.7H₂O,0.5 mg CuSO₄.5H₂O, 1.0 mg H₃BO₃, 1.0 mg MnSO₄.5H₂O, 7.0 mg ZnSO₄.7H₂O,1.0 mg MoO₃, 4.0 g KCl). The culture was grown in a shaker incubator forfour days at 28° C. and then 20 μl was used to inoculate 2.5 ml of freshHMSM containing 10 mM glyphosate as the only phosphorus source. Aftertwo days, 20 μl was used to inoculate another fresh 2.5 ml culture.After 5 days, 20 μl was used to inoculate a fresh 2.5 ml culture. Aftersufficient growth, the culture was plated onto solid media by streakinga 1 μl loop onto the surface of agar plate containing HMSM agarcontaining 10 mM glyphosate as the sole phosphorus source and stored at28° C. The culture was then replated for isolation. One particularstrain, designated ATX20019, was selected due to its ability to grow inthe presence of high glyphosate concentrations. ATX 20019 was determinedto be a member of Ochrobactrum sp./Brucella sp. by sequencing of the 16SrDNA and comparison against a database.

Example 9 Preparation and Screening of Cosmid Libraries

Total DNA was extracted from a culture of ATX20019 using methodscommonly known in the art. The DNA was partially digested withrestriction enzyme Sau3A1 and ligated with SuperCos (Stratagene) vectorfragment according to the manufacturer's directions. Ligation productswere packaged into phage particles using GigaPack III XL packagingextract (Stratagene), transfected into E. coli aroA-cells. E. coli aroA-is a strain in which the native aroA gene, encoding EPSP synthase, hasbeen deleted. This strain cannot grow on M63 medium because it requiresexogenously supplied aromatic amino acids. The presence of a cosmidwhich contains an EPSP synthase gene can genetically complement thearoA-phenotype, that is, allow the strain to grow on M63 medium withoutexogenously supplied aromatic amino acids.

The transfected cells were plated on M63 agar medium containing 50 μg/mlkanamycin M63 agar medium containing 100 mM KH₂PO₄, 15 mM (NH₄)₂SO₄, 50μM CaCl₂, 1 μM FeSO₄, 50 μM MgCl₂, 55 mM glucose, 25 mg/liter L-proline,10 mg/liter thiamine HCl, sufficient NaOH to adjust the pH to 7.0, and15 g/liter agar. Two colonies which grew on this medium were identified.Cosmid DNA was prepared from each of these colonies and re-transformedinto E. coli aroA-cells. In each case, cells retransformed with cosmidDNA grew on M63 medium in the presence of 0 or 10 mM glyphosate whilecells containing empty SuperCos vector did not. This confirms that thecosmids are able to complement the aroA-phenotype and able to conferresistance to glyphosate. These cosmids were named pAX1100 and pAX1101.The cosmids appeared to be identical by restriction digest analysisusing two different enzymes. One cosmid, pAX1101, was selected forfurther characterization.

Example 10 Identification of grg12 in Cosmid pAX1101

To identify the gene(s) responsible for the glyphosate-resistance shownby cosmid pAX1101, DNA from this clone was mutagenized with transposableelements. In this method, one identifies clones that have sufferedtransposon insertions, and have lost the ability to confer glyphosateresistance. The location of the transposon insertions identifies theopen reading frame responsible for the glyphosate resistance phenotype.

Cosmid pAX1101 was subjected to in vitro transposon mutagenesis using anEZ::TN Insertion Kit (Epicentre, Madison, Wis.) and the manufacturer'sprotocol. This process randomly inserts a transposon fragment into thecosmid DNA and thus randomly disrupts the function of genes in thecosmid. The transposons contain a gene encoding resistance to anantibiotic, so transposon insertion clones may be selected by theability to grow in the presence of that antibiotic. The locations of thetransposon insertions may be determined by restriction fragment mappingor by sequencing with primers which anneal in the transposon.

Transposon insertion clones of pAX1101 were transformed into E. colistrain DH5a and plated on M63 medium containing glyphosate. Multipleclones were found which had lost the ability to grow in the presence ofglyphosate, indicating that the transposon had disrupted the generesponsible for resistance.

The DNA sequence was determined for the region of pAX1101 containing thetransposon insertions using sequencing methods well known in the art andis presented as SEQ ID NO:9. An open reading frame (ORF) was identifiedat bases 46 through 1380 of SEQ ID NO:9. This nucleotide sequence isprovided as SEQ ID NO:57, and the corresponding amino acid sequence isprovided as SEQ ID NO:58. Analysis of sequence data from eighttransposon insertion picks that had lost resistance to glyphosaterevealed that all were within the ORF. This indicates that the ORFencodes the resistance to glyphosate conferred by the cosmid. This genewas named grg12. Cosmid pAX1101 containing the grg12 ORF was depositedat the Agricultural Research Service Culture Collection (NRRL) Apr. 4,2005, and assigned Accession No. B-30833.

Example 11 Homology of GRG12 with Other Proteins

GRG12 has homology to EPSP synthase enzymes. An alignment of the GRG12amino acid sequence (SEQ ID NO:10) with the amino acid sequences ofother EPSP synthases is shown in FIG. 1. Table 3 lists the percentidentity of GRG12 to various EPSP synthases. Examination of the aminoacid sequence (SEQ ID NO:10) revealed that it does not contain the fourdomains typical of Class II EPSP synthase enzymes. Thus it is a novel,non-Class II, glyphosate-resistant EPSP synthase.

GRG12 has highest amino acid homology to an EPSPS described inWO2005014820 (SEQ ID NO:54). GRG12 (SEQ ID NO:10) has 92% amino acididentity to the EPSPS described in WO2005014820.

TABLE 3 Amino Acid Identity of GRG12 to Other EPSP Synthases PercentIdentity to Gene GRG12 GRG8 61% GRG7 65% GRG6 65% GRG9 60% D.psychrophila 32% K. pneumoniae 31% E. coli 30% H. influenzae 30% A.fulgidus 22% CP4 22% A. pernix 21% C. perfringens 21% B. subtilis 17%WO2005014820 92%

Further analysis of the grg12 DNA sequence (SEQ ID NO:9) revealed thepresence of a second, shorter open reading frame (SEQ ID NO:58)beginning with a GTG start codon at nucleotide 142 of SEQ ID NO:9.Translation of this open reading frame results in a protein (SEQ IDNO:59) that is identical to residues 33-444 of SEQ ID NO:10, except thatthe start codon of SEQ ID NO:59 is a methionine instead of the valinepresent in SEQ ID NO:10. Alignment of SEQ ID NO:59 with known EPSPSproteins indicated that this protein contains all residues known to becritical to function as an EPSPS. Thus, this protein is likely tocomprise a functional, glyphosate-resistant EPSPS enzyme, whereas aprotein resulting from initiation of translation from a start codoninternal to the highly conserved domains would be unlikely to befunctional. SEQ ID NO:59 is 97% identical to the EPSPS described inW02005014820 (SEQ ID NO:54).

The ability of both open reading frames (SEQ ID NOS:57 and 58) to encodefunctional EPSPS activity may be tested by amplifying each open readingframe by PCR, cloning the resulting PCR fragments into a plasmid vectorunder the control of a suitable promoter, inducing expression of proteinfrom the open reading frame as known in the art, and comparing theability of the expressed protein to complement the aroA-phenotype in E.coli and to confer resistance to glyphosate.

Example 12 Engineering of grg12 for Expression of GRG12 Protein in E.coli

The grg12 open reading frame (ORF) was amplified by PCR, cloned into aslightly modified version of the plasmid vector pUC18 and transformedinto E. coli strain DH5a. The modifications to pUC18 were as follows: astop codon was inserted into the lacZ open reading frame and a ribosomebinding site (to optimize translational initiation of the inserted grg12ORF) was inserted upstream of the BamHI restriction site. Plasmid DNAwas prepared and the presence and orientation of the grg12 insert wasdetermined by restriction digest. One clone which contained the ORF inthe forward orientation with respect to the lac promoter in the vectorwas named pAX1106 and was tested for the ability to confer resistance toglyphosate. E. coli cells harboring pAX1106 or empty vector plasmid werestreaked onto M63 agar plates containing 0 to 200 mM glyphosate. Theresults are presented in Table 4 below. These results demonstrate thatgrg12 confers resistance to high levels of glyphosate.

TABLE 4 Growth of pAX1106 on M63 Glyphosate Agar GlyphosateConcentration (mM) pAX1106 Empty Vector 0 + + 50 + − 100 + − 200 + −

Example 13 Purification of GRG12 Expressed as a 6× His-tagged Protein inE. coli

The grg12 coding region is amplified by PCR using PFUULTRA™ DNApolymerase (Stratagene). Oligonucleotides used to prime PCR are designedto introduce restriction enzyme recognition sites near the 5′ and 3′ends of the resulting PCR product. The resulting PCR product is digestedwith appropriate restriction enzymes and the digested product is clonedinto the 6× His-tag expression vector pRSF1b (Novagen). The resultingclone contains grg12 in the same translational reading frame as, andimmediately C-terminal to, the 6× His tag. General strategies forgenerating such clones, and for expressing proteins containing 6×His-tag are well known in the art. The level of expression of GRG12protein may be determined on an SDS-PAGE protein gel. GRG12 protein canbe isolated by purification of the induced GRG12 protein bychromatography on, for example, Ni-NTA Superflow Resin (Qiagen), as permanufacturer's instructions.

Example 14 Isolation of ATX4150

ATX4150 was isolated by plating samples of soil on Enriched MinimalMedia (EMM) containing glyphosate as the sole source of phosphorus.Since EMM contains no aromatic amino acids, a strain must be resistantto glyphosate in order to grow on this media.

Two grams of soil were suspended in approximately 30 ml of water, andsonicated for 30 seconds in an Aquasonic sonicator water bath. Thesample was vortexed for 5 seconds and permitted to settle for 60seconds. This process was repeated 3 times. 100 μl of this suspensionwas added to 3 ml of EMM supplemented with 4 mM glyphosate (pH 6.0). EMMcontains (per 900 mls): 10 g sucrose, 2 g NaNO₃, 1.0 ml 0.8 M MgSO₄, 1.0ml 0.1 M CaCl₂, 1.0 ml Trace Elements Solution (In 100 ml of 1000×solution: 0.1 g FeSO₄.7H₂O, 0.5 mg CuSO₄.5H₂O, 1.0 mg H₃BO₃, 1.0 mgMnSO₄.5H₂O, 7.0 mg ZnSO₄.7H₂O, 1.0 mg MoO₃, 4.0 g KCl). The culture wasshaken on a tissue culture roller drum for sixteen days at 21° C. andthen 100 μl was used to inoculate 3 ml of fresh EMM containing 4 mMglyphosate as the only phosphorus source. After five days, 100 μl wasused to inoculate another fresh 3 ml culture. After a few days, theculture was plated onto solid media by streaking a 1 μl loop onto thesurface of agar plate containing EMM agar containing 5 mM glyphosate asthe sole phosphorus source. After a few days, colonies were replated forisolation onto EMM containing 5 mM glyphosate as the sole phosphorussource. One particular strain, designated ATX4150, was selected due toits ability to grow in the presence of high glyphosate concentrations.

Example 15 Preparation and Screening of Cosmid Libraries

Total DNA was extracted from a culture of ATX4150 using methods commonlyknown in the art. The DNA was partially digested with restriction enzymeSau3A1 and ligated with SuperCos (Stratagene) vector fragment accordingto the manufacturer's directions. Ligation products were packaged intophage particles using GigaPack III XL packaging extract (Stratagene),transfected into E. coli aroA-cells E. coli aroA-, is a strain in whichthe native aroA gene, encoding EPSP synthase, has been deleted. Thisstrain cannot grow on M63 medium because it requires exogenouslysupplied aromatic amino acids. The presence of a cosmid which containsan EPSP synthase gene can genetically complement the aroA-phenotype,that is, it allow the strain to grow on M63 medium without exogenouslysupplied aromatic amino acids.

The transfected cells were plated on M63 agar medium containing 50 μg/mlkanamycin M63 agar medium contains 100 mM KH₂PO₄, 15 mM (NH₄)₂SO₄, 50 μMCaCl₂, 1 μM FeSO₄, 50 μM MgCl₂, 55 mM glucose, 25 mg/liter L-proline, 10mg/liter thiamine HCl, sufficient NaOH to adjust the pH to 7.0, and 15g/liter agar. Five colonies which grew on this medium were identified.Cosmid DNA was prepared from each of these colonies and re-transformedinto E. coli aroA-cells. In each case cells retransformed with cosmidDNA grew on M63 medium in the presence of 0 or 10 mM glyphosate whilecells containing empty SuperCos vector did not. This confirms that thecosmids are able to complement the aroA-phenotype and able to conferresistance to glyphosate. One cosmid was selected for furthercharacterization and was named pAX305.

Example 16 Identification of grg15 in Cosmid pAX305

To identify the gene(s) responsible for the glyphosate-resistance shownby cosmid pAX305, DNA from this clone was mutagenized with transposableelements. Cosmid pAX305 was subjected to in vitro transposon mutagenesisusing an EZ::TN Insertion Kit (Epicentre, Madison, Wis.) and themanufacturer's protocol. Transposon insertion clones of pAX305 weretransformed into E. coli and plated on M63 medium containing glyphosate.Multiple clones were found which had lost the ability to grow in thepresence of glyphosate, indicating that the transposon had disrupted thegene responsible for resistance.

The DNA sequence was determined for the region of pAX305 containing thetransposon insertions using sequencing methods well known in the art andis presented in SEQ ID NO:61. An open reading frame (ORF, nucleotidebases 77 through 1354 of SEQ ID NO:61) was identified. Analysis ofsequence data from eight transposon insertion picks that had lostresistance to glyphosate revealed that all were within the ORF. Thisindicates that the ORF encodes the resistance to glyphosate conferred bythe cosmid. This gene was named grg15. Cosmid pAX305 containing thegrg15 ORF (SEQ ID NO:11) was deposited at the Agricultural ResearchService Culture Collection (NRRL) on Apr. 20, 2005 and assignedAccession No. NRRL B-30838.

Example 17 Homology of GRG15 with Other Proteins

GRG15 has homology to EPSP synthase enzymes. An alignment of the GRG15amino acid sequence (SEQ ID NO:12) with the amino acid sequences ofother EPSP synthases is shown in FIG. 1. Table 5 lists the percentidentity of GRG15 to various EPSP synthases. Examination of the aminoacid sequence (SEQ ID NO:12) revealed that it does not contain the fourdomains typical of Class II EPSP synthase enzymes. Thus it is a novel,non-Class II, glyphosate-resistant EPSP synthase.

TABLE 5 Amino Acid Identity of GRG15 to Other EPSP Synthases PercentIdentity to Gene GRG15 GRG9 94% GRG8 70% GRG7 66% GRG6 66% GRG12 60% K.pneumoniae 32% E. coli 32% H. influenzae 32% D. psychrophila 31% A.fulgidus 26% A. pernix 26% C. perfringens 24% B. subtilis 24% GRG1 23%Agrobacterium Sp. 22% CP4

Example 18 Homology Blocks among Glyphosate Resistant Non-Class IIEnzymes (Class III)

Comparison of the amino acid sequences of the GRG proteins (non-class IIglyphosate resistant proteins) show that the GRG proteins havesignificant homology to one another, and are distinct from previouslyidentified glyphosate resistant EPSP synthases (see FIG. 1).

Example 19 Identification of Conserved Domains

The amino acid sequences of seven known EPSP synthases were analyzed forconserved domains that were not present in either class I or class IIEPSP synthases. The following domains are found in these EPSP synthases:Bold type denotes conserved residues only among this class, and italicsdenote residues conserved in all EPSPS enzymes.

Domain I:

-   L-A-K-G-X₁-S-X₂-L-X₃-G-A-L-K-S-D-D-T (SEQ ID NO:13), where X₁    denotes lysine or threonine, where X₂ denotes arginine or histidine,    where X₃ denotes serine or threonine.

Domain Ia:

-   L-A-K-G-X₁, (SEQ ID NO:14), where X₁ denotes lysine or threonine.

Domain Ib:

-   S-X₁-L-X₂(SEQ ID NO:15), where X₁ denotes arginine or histidine,    where X₂ denotes serine or threonine.

Domain Ic:

-   G-A-L-K-S-D-D-T (SEQ ID NO:16)

Domain II:

-   E-P-D-X₁-X₂-T-F-X₃-V-X₄-X₅-X₆-G (SEQ ID NO:17), where X₁ denotes    aspartic acid or alanine, where X₂ denotes serine or threonine,    where X₃ denotes valine or isoleucine, where X₄ denotes threonine or    glutamic acid or lysine, where X₅ denotes serine or glycine, where    X₆ denotes glutamine or serine or glutamic acid or threonine.

Domain IIa:

-   E-P-D-X₁-X₂-T-F-X₃-V (SEQ ID NO:18), where X₁ denotes aspartic acid    or alanine, where X₂ denotes serine or threonine, where X₃ denotes    valine or isoleucine.

Domain IIb:

-   X₁-X₂-X₃-G (SEQ ID NO:19), where X₁ denotes threonine or glutamic    acid or lysine, where X₂ denotes serine or glycine, where X₃ denotes    glutamine or serine or glutamic acid or threonine.

Domain III:

-   RFLTAA (SEQ ID NO:20)

Domain IV:

-   KRPI(G/M)P (SEQ ID NO:21)-   K-R-P-I-X₁-P, where X₁ denotes glycine or methionine or leucine.

Domain V:

-   X₁-G-C-P-P-V (SEQ ID NO:22), where X₁ denotes threonine or serine.

Domain VI:

-   I-G-A-X₁-G-Y-X₂-D-L-T (SEQ ID NO:23), where X₁ denotes arginine or    lysine or leucine, and where X₂ denotes isoleucine or valine.

Domain VII:

-   W-X₁-V-X₂-X₃-T-G (SEQ ID NO:24), where X₁ denotes arginine or    lysine, where X₂ denotes alanine or histidine or glutamic acid or    serine, where X₃ denotes proline or alanine

Domain VIII:

-   E-P-D-A-S-A-A-T-Y-L-W-X₁-A-X₂-X₃-L (SEQ ID NO:25), where X₁ denotes    alanine or glycine, where X₂ denotes glutamic acid or glutamine,    where X₃ denotes valine or leucine or alanine

Domain VIIIa:

-   E-P-D-A-S-A-A-T-Y-L-W (SEQ ID NO:26)

Domain IX:

-   I-D-X₁-G (SEQ ID NO:27), where X₁ denotes isoleucine or leucine.

Domain X:

-   F-X₁-Q-P-D-A-K-A (SEQ ID NO:28), where X₁ denotes threonine or    serine,

Domain XI:

-   X₁-F-P-X₂-X₃-X₄-A-X₅-X₆-X₇-G-S-Q-M-Q-D-A-I-P-T-X₈-A-V-X₉A-A-F-N (SEQ    ID NO:29), where X₁ denotes glutamine or lysine or serine, where X₂    denotes asparagine or histidine, where X₃ denotes methionine or    leucine, where X₄ denotes proline or glutamine, where X₅ denotes    threonine or glutamic acid or valine, where X₆ denotes valine or    isoleucine, where X₇ denotes aspartic acid or valine, where X₈    denotes leucine or isoleucine, where X₉ denotes leucine or    isoleucine.

Domain XIa:

-   X₁-F-P-X₂-X₃-X₄-A (SEQ ID NO:30), where X₁ denotes glutamine or    lysine or serine, where X₂ denotes asparagine or histidine, where X₃    denotes methionine or leucine, where X₄ denotes proline or    glutamine.

Domain XIb:

-   X₁-X₂-X₃-G-S-Q-M-Q-D-A-I-P-T-X₄-A-V-X₅A-A F-N (SEQ ID NO:31), where    X₁ denotes threonine or glutamic acid or valine, where X₂ denotes    valine or isoleucine, where X₃ denotes aspartic acid or valine,    where X₄ denotes leucine or isoleucine, where X₅ denotes leucine or    isoleucine.

Domain XIc:

-   G-S-Q-M-Q-D-A-I-P-T (SEQ ID NO:32)

Domain XII:

-   P-V-R-F-X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:33), where X₁    denotes valine or threonine, where X₂ denotes glutamic acid or    glycine, where X₃ denotes leucine or isoleucine, where X₄ denotes    alanine or glutamic acid, where X₅ denotes isoleucine or valine.

Domain XIIa:

-   P-V-R-F (SEQ ID NO:34)

Domain XIIb:

-   X₁-X₂-X₃-X₄-N-L-R-V-K-E-C-D-R-X₅ (SEQ ID NO:35), where X₁ denotes    valine or threonine, where X₂ denotes glutamic acid or glycine,    where X₃ denotes leucine or isoleucine, where X₄ denotes alanine or    glutamic acid, where X₅ denotes isoleucine or valine.

Domain XIIc:

-   N-L-R-V-K-E-C-D-R (SEQ ID NO:36)

Domain XIII:

-   E-G-D-D-L-X₁-X₂ (SEQ ID NO:37), where X₁ denotes leucine or    isoleucine, where X₂ denotes valine or isoleucine.

Domain XIV:

-   X₁-P-X₂-L-A-G (SEQ ID NO:38), where X₁ denotes aspartic acid or    asparagine, where X₂ denotes alanine or serine or threonine.

Domain XV:

-   A-X₁-I-D-X₂-X₃-X₄-D-H-R- (SEQ ID NO:39), where X₁ denotes leucine or    serine or glutamic acid, where X₂ denotes threonine or serine, where    X₃ denotes histidine or phenylalanine, where X₄ denotes alanine or    serine.

Domain XVI:

-   F-A-L-A-X₁-L-K-X₂-X₃-G-I (SEQ ID NO:40), where X₁ denotes glycine or    alanine, where X₂ denotes isoleucine or valine, where X₃ denotes    serine or glycine or alanine or lysine.

Domain XVIa:

-   F-A-L-A-X₁-L-K (SEQ ID NO:41), where X₁ denotes glycine or alanine

Domain XVIb:

-   L-K-X₁-X₂-G-I (SEQ ID NO:42), where X₁ denotes isoleucine or valine,    where X₂ denotes serine or glycine or alanine or lysine.

Domain XVII:

-   -X₁-P-X₂-C-V-X₃-K (SEQ ID NO:43), where X₁ denotes asparagine or    aspartic acid, where X₂ denotes alanine or aspartic acid, where X₃    denotes alanine or glycine.

Domain XVIII:

-   X₁-S-L-G-V (SEQ ID NO:44), where X₁ denotes alanine or serine or    proline.

Example 20 Engineering grg12 for Plant Transformation

The grg12 open reading frame (ORF) is amplified by PCR from afull-length cDNA template. Hind III restriction sites are added to eachend of the ORF during PCR.

Additionally, the nucleotide sequence ACC is added immediately 5′ to thestart codon of the gene to increase translational efficiency (Kozak(1987) Nucleic Acids Research 15:8125-8148; Joshi (1987) Nucleic AcidsResearch 15:6643-6653). The PCR product is cloned and sequenced, usingtechniques well known in the art, to ensure that no mutations areintroduced during PCR.

The plasmid containing the grg12 PCR product is digested with, forexample, Hind III and the fragment containing the intact ORF isisolated. In this example, the fragment is cloned into the Hind III siteof a plasmid, such as pAX200, which is a plant expression vectorcontaining the rice actin promoter (McElroy et al. (1991) Molec. Gen.Genet. 231:150-160), and the PinII terminator (An et al. (1989) ThePlant Cell 1:115-122). The promoter—gene—terminator fragment from thisintermediate plasmid is then subcloned into a plasmid such as pSB11(Japan Tobacco, Inc.) to form a final plasmid, referred to herein aspSB11GRG12. pSB11GRG12 is organized such that the DNA fragmentcontaining the promoter—grg12—terminator construct may be excised byappropriate restriction enzymes and also used for transformation intoplants, for example, by aerosol beam injection. The structure ofpSB11GRG12 is verified by restriction digests and gel electrophoresisand by sequencing across the various cloning junctions.

The plasmid is mobilized into Agrobacterium tumefaciens strain LBA4404which also harbors the plasmid pSB1 (Japan Tobacco, Inc.), usingtriparental mating procedures well known in the art, and plating onmedia containing antibiotic. Plasmid pSB11GRG12 carries spectinomycinresistance but is a narrow host range plasmid and cannot replicate inAgrobacterium. Antibiotic resistant colonies arise when pSB11GRG12integrates into the broad host range plasmid pSB1 through homologousrecombination. The resulting cointegrate product is verified by Southernhybridization. The Agrobacterium strain harboring the cointegrate can beused to transform maize, for example, by the PureIntro method (JapanTobacco).

Example 21 Transformation of grg12 into Plant Cells

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to express GRG12 in plant cells are acceleratedinto plant tissue using an aerosol beam accelerator, using conditionsessentially as described in PCT Publication No. WO/0138514. Afterbeaming, embryos are incubated for about 30 min on osmotic media, andplaced onto incubation media overnight at 25° C. in the dark. To avoidunduly damaging beamed explants, they are incubated for at least 24hours prior to transfer to recovery media. Embryos are then spread ontorecovery period media, for about 5 days, 25° C. in the dark, thentransferred to a selection media. Explants are incubated in selectionmedia for up to eight weeks, depending on the nature and characteristicsof the particular selection utilized. After the selection period, theresulting callus is transferred to embryo maturation media, until theformation of mature somatic embryos is observed. The resulting maturesomatic embryos are then placed under low light, and the process ofregeneration is initiated by methods known in the art. The resultingshoots are allowed to root on rooting media, and the resulting plantsare transferred to nursery pots and propagated as transgenic plants.

Materials

TABLE 6 DN62A5S Media Components Per Liter Source Chu's N6 Basal Salt3.98 g/L Phytotechnology Labs Mixture (Prod. No. C 416) Chu's N6 Vitamin1 mL/L (of Phytotechnology Labs Solution (Prod. No. 1000x Stock) C 149)L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100 mg/L SigmaL-Proline 1.4 g/L Phytotechnology Labs Casamino acids 100 mg/L FisherScientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1 mL/L(of Sigma D-7299) 1 mg/mL Stock)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 22 Transformation of grg12 into Maize Plant Cells byAgrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art. The resulting shoots are allowed to rooton rooting media, and the resulting plants are transferred to nurserypots and propagated as transgenic plants.

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A recombinant polynucleotide encoding a glyphosate-tolerant5-enolpyruvylshikimate-3-phosphate (EPSP) synthase enzyme, wherein saidpolynucleotide is selected from the group consisting of: a) thenucleotide sequence of SEQ ID NO:1, 45, 47, and 55; b) a nucleotidesequence that encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO:2, 46, 48, and 56; and, c) a nucleotide sequence encoding apolypeptide having at least 95% amino acid sequence identity to theamino acid sequence of SEQ ID NO:2, 46, 48, and 56, wherein saidnucleotide sequence encodes a glyphosate tolerant EPSP synthase enzyme,wherein said polynucleotide is operably linked to a promoter that drivesexpression of said polynucleotide in a plant cell.
 2. The polynucleotideof claim 1, wherein said promoter is heterologous with respect to thepolynucleotide.
 3. The polynucleotide of claim 1 in which thepolynucleotide encodes a fusion polypeptide comprising an amino-terminalchloroplast transit peptide and the EPSP synthase enzyme.
 4. Aglyphosate-tolerant plant cell comprising the polynucleotide of claim 1.5. The glyphosate-tolerant plant cell of claim 4 selected from the groupconsisting of corn, wheat, rice, barley, soybean, cotton, sugarbeet,oilseed rape, canola, flax, sunflower, potato, tobacco, tomato, alfalfa,poplar, pine, eucalyptus, apple, lettuce, peas, lentils, grape and turfgrasses.
 6. A glyphosate-tolerant plant comprising the plant cell ofclaim
 4. 7. The glyphosate-tolerant plant of claim 6 selected from thegroup consisting of corn, wheat, rice, barley, soybean, cotton,sugarbeet, oilseed rape, canola, flax, sunflower, potato, tobacco,tomato, alfalfa, poplar, pine, eucalyptus, apple, lettuce, peas,lentils, grape and turf grasses.
 8. A transgenic seed comprising thepolynucleotide of claim
 1. 9. A method of producing geneticallytransformed plants which are tolerant toward glyphosate herbicide,comprising the steps of: a) inserting into the genome of a plant cell apolynucleotide comprising a promoter which functions in plant cells tocause the production of an RNA sequence operably linked to a nucleotidesequence encoding a glyphosate-tolerant 5-EPSP synthase enzyme, whereinsaid nucleotide sequence is selected from the group consisting of: i)the nucleotide sequence of SEQ ID NO:1, 45, 47, and 55; ii) a nucleotidesequence that encodes a polypeptide comprising the amino acid sequenceof SEQ ID NO:2, 46, 48, and 56; and, iii) a nucleotide sequence encodinga polypeptide having at least 95% amino acid sequence identity to theamino acid sequence of SEQ ID NO:2, 46, 48, and 56, wherein saidnucleotide sequence encodes a glyphosate-tolerant EPSP synthase enzyme;b) obtaining a transformed plant cell; and c) regenerating from thetransformed plant cell a genetically transformed plant which hasincreased tolerance to glyphosate herbicide.
 10. The method of claim 9,wherein obtaining the transformed plant comprises screening for anenhanced tolerance to glyphosate herbicide.
 11. The method of claim 9 inwhich the polynucleotide encodes a fusion polypeptide comprising anamino-terminal chloroplast transit peptide and the EPSP synthase enzyme.12. A method for selectively controlling weeds in a field having plantedseeds or plants comprising the steps of: a) growing in said field theplant of claim 6 or a transgenic seed obtained therefrom, and b)applying to the plants and weeds in the field a sufficient amount ofglyphosate herbicide to control the weeds without significantlyaffecting the plants.
 13. A method for increasing yield in a plantcomprising growing in a field the plant of claim 6 or a transgenic seedobtained therefrom and applying to the plant an effective concentrationof glyphosate herbicide.